Preparation method of biotinylated protein and detection method using the same

Abstract

The present invention presents construction of a detection method requiring no step of removing free biotin during preparation of a biotinylated protein having a biotin tag, in a detection method of a substance interacting with a protein, and studied various preparation methods of the biotinylated protein. In order to solve the above-mentioned problem, the present inventor has found that in a cell-free protein synthesizing system, in particular, a wheat embryo cell-free protein synthesizing system, when biotinylation is performed during or after protein's synthesis, the biotinylation of the protein can be attained in an remarkably lower concentration of the biotin than that in the conventional biotinylation operations, and has accomplished the present invention by using the protein having the biotin tag in each detection system.

1. A method for detecting a substance interacting with a protein, wherein a protein with a biotin binding sequence is simultaneously translated and biotinylated using a wheat germ cell-free protein synthesis system in the presence of a biotinylating enzyme and a biotin derivative, and a substance interacting with the biotinylated protein is subsequently detected. 
     
     
       2. The detection method of  claim 1 , wherein the substance interacting with the biotinylated protein is detected without removing the biotin derivative which was not bound to the protein. 
     
     
       3. The detection method of  claim 1 , which is at least one selected from 1) amplified luminescence proximity homogenous assay (ALPHA) 2) surface plasmon resonance technique (SPR), 3) fluorescence correlation spectroscopy (FCS), 4) fluorescence intensity distribution analysis (FIDA), 5) enzyme-linked immuno adsorbent assay (ELISA), 6) dissociation enhanced lanthanide fluoro immuno assay (DELFIA), 7) scintillation proximity analysis (SPA), 8) fluorescence resonance energy transfer (FRET), 9) bioluminescent resonance energy transfer (BRET), 10) enzyme fragment complementation (EFC), and 11) fluorescence polarization (FP). 
     
     
       4. The detection method of  claim 2 , which is at least one selected from 1) ALPHA, 2) SPR, 3) FCS, 4) FIDA, 5) ELISA, 6) DELFIA, 7) SPA, 8) FRET, 9) BRET, 10) EFC, and 11)FP. 
     
     
       5. The detection method of  claim 1 , wherein the biotinylated protein is fixed to a carrier through a biotin linkage to detect the substance interacting with the biotinylated protein. 
     
     
       6. The detection method of  claim 2 , wherein the biotinylated protein is fixed to a carrier through a biotin linkage to detect the substance interacting with the biotinylated protein. 
     
     
       7. The detection method of  claim 3 , wherein the biotinylated protein is fixed to a carrier through a biotin linkage to detect the substance interacting with the biotinylated protein. 
     
     
       8. The detection method of  claim 1 , wherein the substance interacting with the biotinylated protein is detected using a labeled biotin derivative as a marker. 
     
     
       9. The detection method of  claim 2 , wherein the substance interacting with the biotinylated protein is detected using a labeled biotin derivative as a marker. 
     
     
       10. The detection method of  claim 3 , wherein the substance interacting with the biotinylated protein is detected using a labeled biotin derivative as a marker. 
     
     
       11. The detection method of  claim 5 , wherein the substance interacting with the biotinylated protein is detected using a labeled biotin derivative as a marker.